Methods for producing polypeptides by regulating polypeptide association

Abstract

In the course of the present invention, it was discovered that one could regulate association between polypeptides by modifying amino acid residues that form the interface during the association to amino acids carrying the same type of charge. In this context, the present invention enables efficient formation of heterologous molecules. For example, the present invention can be suitably applied to the preparation of bispecific antibodies.

1 . A method for producing a polypeptide comprising a mutation in an amino acid residue forming a polypeptide interface such that polypeptide association will be regulated, wherein the method comprises:
 (a) modifying a nucleic acid encoding an amino acid residue forming the polypeptide interface from the original nucleic acid, such that polypeptide association will be inhibited;   (b) culturing host cells such that said nucleic acid is expressed; and   (c) recovering said polypeptide from the host cell culture.   
     
     
         2 . A method for producing a heteromultimer comprising a mutation in an amino acid residue forming an interface between polypeptides such that heteromultimer association will be regulated, wherein the method comprises:
 (a) modifying a nucleic acid encoding an amino acid residue forming the interface between polypeptides from the original nucleic acid, such that the association between polypeptides will be inhibited;   (b) culturing host cells such that said nucleic acid is expressed; and   (c) recovering said heteromultimer from the host cell culture.   
     
     
         3 . The method of  claim 1 , wherein a nucleic acid encoding an amino acid residue forming a polypeptide interface is modified from the original nucleic acid, so that the polypeptide association forming one or more types of conformational isomers will be inhibited in a polypeptide that may form two or more types of conformational isomers. 
     
     
         4 . The method of  claim 2 , wherein a nucleic acid encoding an amino acid residue forming an interface between polypeptides is modified from the original nucleic acid, so that the association between polypeptides forming one or more types of multimers will be inhibited in a heteromultimer that may form two or more types of multimers. 
     
     
         5 . The method of  claim 1 , wherein the modification of step (a) is modifying the original nucleic acid so that an amino acid residue mutation is introduced to the interface such that two or more amino acid residues forming the interface will carry the same type of charge. 
     
     
         6 . The method of  claim 5 , wherein the introduced amino acid residue is glutamic acid (E). 
     
     
         7 . The method of  claim 5 , wherein the introduced amino acid residue is aspartic acid (D). 
     
     
         8 . The method of  claim 5 , wherein the introduced amino acid residue is lysine (K). 
     
     
         9 . The method of  claim 5 , wherein the introduced amino acid residue is arginine (R). 
     
     
         10 . The method of  claim 5 , wherein the introduced amino acid residue is histidine (H). 
     
     
         11 . The method of  claim 1 , wherein the modification of step (a) is modifying the original nucleic acid so that an amino acid residue mutation is introduced to the interface such that an amino acid residue forming a hydrophobic core present in the interface will become charged amino acid residues. 
     
     
         12 . The method of  claim 11 , wherein the introduced amino acid residue is glutamic acid (E). 
     
     
         13 . The method of  claim 11 , wherein the introduced amino acid residue is aspartic acid (D). 
     
     
         14 . The method of  claim 11 , wherein the introduced amino acid residue is lysine (K). 
     
     
         15 . The method of  claim 11 , wherein the introduced amino acid residue is arginine (R). 
     
     
         16 . The method of  claim 11 , wherein the introduced amino acid residue is histidine (H). 
     
     
         17 . The method of  claim 1 , wherein the interface of the polypeptide is formed by an antibody heavy chain variable region and light chain variable region. 
     
     
         18 . The method of  claim 1 , wherein the polypeptide interface is formed by two or more types of heavy chain variable regions. 
     
     
         19 . The method of  claim 1 , wherein the polypeptide interface is formed by an antibody heavy chain constant region and light chain constant region. 
     
     
         20 . The method of  claim 1 , wherein the polypeptide interface is formed by two or more types of heavy chain constant regions. 
     
     
         21 . The method of  claim 1 , wherein the polypeptide is a single chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are linked by linkers. 
     
     
         22 . The method of  claim 2 , wherein the heteromultimer is a multi-specific antibody comprising two or more types of heavy chain variable regions and two or more types of light chain variable regions. 
     
     
         23 . The method of  claim 22 , wherein the heteromultimer is a bispecific antibody. 
     
     
         24 . A mutant polypeptide or heteromultimer produced by the method of  claim 1 . 
     
     
         25 . A mutant polypeptide, comprising a modification made to an amino acid residue forming an interface in the original polypeptide such that the association within said polypeptide is inhibited. 
     
     
         26 . A heteromultimer, comprising a modification made to an amino acid residue forming an interface between the original polypeptides such that the association between said polypeptides is inhibited. 
     
     
         27 . The mutant polypeptide of  claim 25 , wherein the original polypeptide may form two or more types of conformational isomers. 
     
     
         28 . The heteromultimer of  claim 26 , wherein the original polypeptides may form two or more types of multimers. 
     
     
         29 . The mutant polypeptide of  claim 25 , wherein said modification of the amino acid residues forming a polypeptide interface is introducing an amino acid residue mutation to the interface such that two or more amino acid residues forming the interface will carry the same type of charge. 
     
     
         30 . The mutant polypeptide or heteromultimer of  claim 29 , wherein the introduced amino acid residue is glutamic acid (E). 
     
     
         31 . The mutant polypeptide or heteromultimer of  claim 29 , wherein the introduced amino acid residue is aspartic acid (D). 
     
     
         32 . The mutant polypeptide or heteromultimer of  claim 29 , wherein the introduced amino acid residue is lysine (K). 
     
     
         33 . The mutant polypeptide or heteromultimer of  claim 29 , wherein the introduced amino acid residue is arginine (R). 
     
     
         34 . The mutant polypeptide or heteromultimer of  claim 29 , wherein the introduced amino acid residue is histidine (H). 
     
     
         35 . The mutant polypeptide of  claim 25 , wherein the modification of amino acid residues forming the polypeptide interface is introducing an amino acid residue mutation to the interface such that an amino acid residue forming a hydrophobic core present in the interface will become charged amino acid residues. 
     
     
         36 . The mutant polypeptide or heteromultimer of  claim 35 , wherein the introduced amino acid residue is glutamic acid (E). 
     
     
         37 . The mutant polypeptide or heteromultimer of  claim 35 , wherein the introduced amino acid residue is aspartic acid (D). 
     
     
         38 . The mutant polypeptide or heteromultimer of  claim 35 , wherein the introduced amino acid residue is lysine (K). 
     
     
         39 . The mutant polypeptide or heteromultimer of  claim 35 , wherein the introduced amino acid residue is arginine (R). 
     
     
         40 . The mutant polypeptide or heteromultimer of  claim 35 , wherein the introduced amino acid residue is histidine (H). 
     
     
         41 . The mutant polypeptide of  claim 25 , wherein the polypeptide interface is formed by an antibody heavy chain variable region and light chain variable region. 
     
     
         42 . The mutant polypeptide of  claim 25 , wherein the polypeptide interface is formed by two or more types of heavy chain variable regions. 
     
     
         43 . The mutant polypeptide of  claim 25 , wherein the polypeptide interface is formed by an antibody heavy chain constant region and light chain constant region. 
     
     
         44 . The mutant polypeptide of  claim 25 , wherein the polypeptide interface is formed by two of more types of heavy chain constant regions. 
     
     
         45 . The mutant polypeptide of  claim 25 , wherein the polypeptide is a single chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are linked by linkers. 
     
     
         46 . The heteromultimer of  claim 26 , wherein the heteromultimer is a multi specific antibody comprising two or more types of heavy chain variable regions and two or more types of light chain variable regions. 
     
     
         47 . The heteromultimer of  claim 46 , wherein the heteromultimer is a bispecific antibody. 
     
     
         48 . A composition comprising the mutant polypeptide of  claim 25 , and a pharmaceutically acceptable carrier. 
     
     
         49 . A nucleic acid encoding the mutant polypeptide of  claim 25 . 
     
     
         50 . A host cell comprising the nucleic acid of  claim 49 . 
     
     
         51 . A method for producing the mutant polypeptide of  claim 25 , which comprises the steps of culturing the host cell, and recovering the polypeptide from the cell culture. 
     
     
         52 . A method for regulating polypeptide association, which comprises modifying an amino acid residue forming an interface in the original polypeptide such that the association within the polypeptide is inhibited. 
     
     
         53 . A method for regulating heteromultimer association, which comprises modifying amino acid residues forming an interface between the original polypeptides such that the association between the polypeptides is inhibited. 
     
     
         54 . The method of  claim 52 , which comprises modifying an amino acid residue forming an interface in a polypeptide, such that the association of a polypeptide forming one or more types of conformational isomers will be inhibited in a polypeptide that may form two or more types of conformational isomers. 
     
     
         55 . The method of  claim 53 , which comprises modifying amino acid residues forming an interface between polypeptides, such that the association between polypeptides that form one or more types of conformational isomers will be inhibited in a heteromultimer that may form two or more types of multimers. 
     
     
         56 . The method of  claim 52 , wherein said modification of an amino acid residue forming a polypeptide interface is an amino acid residue mutation to the interface such that two or more amino acid residues forming the interface will have the same type of charge. 
     
     
         57 . The method of  claim 56 , wherein the introduced amino acid residue is glutamic acid (E). 
     
     
         58 . The method of  claim 56 , wherein the introduced amino acid residue is aspartic acid (D). 
     
     
         59 . The method of  claim 56 , wherein the introduced amino acid residue is lysine (K). 
     
     
         60 . The method of  claim 56 , wherein the introduced amino acid residue is arginine (R). 
     
     
         61 . The method of  claim 56 , wherein the introduced amino acid residue is histidine (H). 
     
     
         62 . The method of  claim 52 , wherein said modification of amino acid residues forming a polypeptide interface is introducing an amino acid residue mutation to the interface such that an amino acid residue forming a hydrophobic core present in the interface will become charged amino acid residues. 
     
     
         63 . The method of  claim 62 , wherein the introduced amino acid residue is glutamic acid (E). 
     
     
         64 . The method of  claim 62 , wherein the introduced amino acid residue is aspartic acid (D). 
     
     
         65 . The method of  claim 62 , wherein the introduced amino acid residue is lysine (K). 
     
     
         66 . The method of  claim 62 , wherein the introduced amino acid residue is arginine (R). 
     
     
         67 . The method of  claim 62 , wherein the introduced amino acid residue is histidine (H). 
     
     
         68 . The method of  claim 52 , wherein the polypeptide interface is formed by an antibody heavy chain variable region and light chain variable region. 
     
     
         69 . The method of  claim 52 , wherein the polypeptide interface is formed by two or more types of heavy chain variable regions. 
     
     
         70 . The method of  claim 52 , wherein the polypeptide interface is formed by an antibody heavy chain constant region and light chain constant region. 
     
     
         71 . The method of  claim 52 , wherein the polypeptide interface is formed by two or more types of heavy chain constant regions. 
     
     
         72 . The method of  claim 52 , wherein the polypeptide is a single chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are linked by linkers. 
     
     
         73 . The method of  claim 53 , wherein the heteromultimer is a multispecific antibody comprising two types or more of heavy chain variable regions and two types or more of light chain variable regions. 
     
     
         74 . The method of  claim 73 , wherein the heteromultimer is a bispecific antibody. 
     
     
         75 . An antibody comprising a heavy chain variable region and a light chain variable region, wherein the following amino acid residues of (1) and (2) carry the same type of charge:
 (1) an amino acid residue which is included in the heavy chain variable region and corresponds to position 39 in the amino acid sequence of SEQ ID NO: 6; and   (2) an amino acid residue which is included in the light chain variable region and corresponds to position 44 in the amino acid sequence of SEQ ID NO: 8.   
     
     
         76 . An antibody comprising a heavy chain variable region and a light chain variable region, wherein the following amino acid residues of (1) and (2) carry the same type of charge:
 (1) an amino acid residue which is included in the heavy chain variable region and corresponds to position 45 in the amino acid sequence of SEQ ID NO: 6; and   (2) an amino acid residue which is included in the light chain variable region and corresponds to position 50 in the amino acid sequence of SEQ ID NO: 8.   
     
     
         77 . An antibody comprising a heavy chain variable region and a light chain variable region, wherein either one of the following amino acid residues of (1) or (2) is a charged amino acid residue:
 (1) an amino acid residue which is included in the heavy chain variable region and corresponds to position 45 in the amino acid sequence of SEQ ID NO: 6; and   (2) an amino acid residue which is included in the light chain variable region and corresponds to position 50 in the amino acid sequence of SEQ ID NO: 8.   
     
     
         78 . The antibody of  claim 75 , wherein amino acid residues carrying the same type of charge are selected from amino acid residues included in the group of either (a) or (b):
 (a) glutamic acid (E) and aspartic acid (D); or   (b) lysine (K), arginine (R), and histidine (H).   
     
     
         79 . The antibody of  claim 77 , wherein said charged amino acid residue is glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), or histidine (H). 
     
     
         80 . The antibody of  claim 75 , wherein the polypeptide is a single chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are linked by linkers. 
     
     
         81 . The antibody of  claim 75 , wherein the polypeptide is a multispecific antibody comprising two or more types of heavy chain variable regions and two or more types of light chain variable regions. 
     
     
         82 . The antibody of  claim 81 , wherein the polypeptide is a bispecific antibody. 
     
     
         83 . A composition comprising the antibody of  claim 75  and a pharmaceutically acceptable carrier. 
     
     
         84 . A nucleic acid encoding a polypeptide constituting the antibody of  claim 75 . 
     
     
         85 . A host cell comprising the nucleic acid of  claim 84 . 
     
     
         86 . The method for producing the antibodies of  claim 75 , which comprises the steps of culturing the host cell and recovering the polypeptides from the cell culture. 
     
     
         87 . An antibody comprising two or more types of heavy chain CH3 regions, wherein one to three pair(s) of amino acid residues in the first heavy chain CH3 region is/are selected from the pair(s) of amino acid residues indicated in (1) to (3) that carry the same type of charge:
 (1) amino acid residues included in the heavy chain CH3 region at positions 356 and 439 according to the EU numbering system;   (2) amino acid residues included in the heavy chain CH3 region at positions 357 and 370 according to the EU numbering system; and   (3) amino acid residues included in the heavy chain CH3 region at positions 399 and 409 according to the EU numbering system.   
     
     
         88 . The antibody of  claim 87 , in which pairs of the amino acid residues in the second heavy chain CH3 region are selected from the pairs of amino acid residues of (1) to (3), wherein the one to three pairs of amino acid residues corresponding to the pairs of amino acid residues of (1) to (3) carrying the same type of charge in said first heavy chain CH3 region, carry opposite charges from the corresponding amino acid residues in said first heavy chain CH3 region. 
     
     
         89 . The antibody of  claim 87 , wherein said amino acid residues carrying the same type of charge are selected from the amino acid residues included in the group of either (a) or (b):
 (a) glutamic acid (E) and aspartic acid (D); or   (b) lysine (K), arginine (R), and histidine (H).   
     
     
         90 . The antibody of  claim 87 , wherein said first heavy chain CH3 region and the second heavy chain CH3 region are crosslinked by a disulfide bond. 
     
     
         91 . The antibody of  claim 87 , wherein the antibody comprises two or more types of heavy chain constant regions. 
     
     
         92 . The antibody of  claim 87 , wherein the multispecific antibody comprises two or more types of heavy chain variable regions and two or more types of light chain variable regions. 
     
     
         93 . The antibody of  claim 92 , which is a bispecific antibody. 
     
     
         94 . A composition comprising the antibody of  claim 87  and a pharmaceutically acceptable carrier. 
     
     
         95 . A nucleic acid encoding a polypeptide constituting the antibody of  claim 87 . 
     
     
         96 . A host cell comprising the nucleic acid of  claim 95 . 
     
     
         97 . A method for producing the antibody of  claim 87 , which comprises the steps of culturing the host cell of  claim 96 , and recovering the polypeptides from the cell culture.